Stem cells are undifferentiated, or immature, cells that are capable of giving rise to multiple specialized cell types and, ultimately, to terminally differentiated cells. Most adult stem cells are lineage-restricted and are generally referred to by their tissue origin. Unlike any other cells, stem cells are able to renew themselves such that a virtually endless supply of mature cell types can be generated when needed over the lifetime of an organism. Due to this capacity for self-renewal, stem cells are therapeutically useful for the formation, regeneration, repair and maintenance of tissues. To ensure self-renewal, stem cells undergo two types of cell division. Symmetric division gives rise to two identical daughter cells, both endowed with stem cell properties, and leads to expansion of the stem cell population. Asymmetric division, on the other hand, produces one stem cell and one progenitor cell with limited self-renewal potential. Progenitors are transient amplifying cells that can go through several rounds of cell division, i.e. proliferation, before terminally differentiating into a mature cell. In adult organisms, stem cells and progenitor cells act as a repair system for the tissues of the body, replenish specialized cells, and maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
It has recently been determined that satellite cells represent a heterogeneous population composed of stem cells and small mononuclear progenitor cells found in mature muscle tissue (Kuang et al., 2007). Satellite cells in adult skeletal muscle are located in small depressions between the sarcolemma of their host myofibers and the basal lamina. Satellite cells are involved in the normal growth of muscle, as well as the regeneration of injured or diseased tissue. In undamaged muscle, the majority of satellite cells are quiescent, meaning they neither differentiate nor undergo cell division. Satellite cells express a number of distinctive genetic markers, including the paired-box transcription factor Pax7, which plays a central regulatory role in satellite cell function and survival (Kuang et al., 2006; Seale et al., 2000). Pax7 can thus be used as a marker of satellite cells.
Upon damage, such as physical trauma or strain, repeated exercise, or in disease, satellite cells become activated, proliferate and give rise to a population of transient amplifying progenitors, which are myogenic precursors cells (myoblasts) expressing myogenic regulatory factors (MRF), such as MyoD and Myf5. In the course of the regeneration process, myoblasts undergo multiple rounds of division before committing to terminal differentiation, fusing with the host fibers or generating new myofibers to reconstruct damaged tissue (Charge and Rudnicki, 2004). In several diseases and conditions affecting muscle, a reduction in muscle mass is seen that is associated with reduced numbers of satellite cells and a reduced ability of the satellite cells to repair, regenerate and grow skeletal muscle. A few exemplary diseases and conditions affecting muscle include wasting diseases, such as cachexia, muscular attenuation or atrophy, including sarcopenia, ICU-induced weakness, surgery-induced weakness (e.g. following knee or hip replacement), and muscle degenerative diseases, such as muscular dystrophies. The process of muscle regeneration involves considerable remodeling of extracellular matrix and, where extensive damage occurs, is incomplete. Fibroblasts within the muscle deposit scar tissue, which can impair muscle function, and is a significant part of the pathology of muscular dystrophies.
Muscular dystrophies are genetic diseases characterized by progressive weakness and degeneration of the skeletal or voluntary muscles which control movement. The muscles of the heart and some other involuntary muscles are also affected in some forms of muscular dystrophy. In many cases, the histological picture shows variation in fiber size, muscle cell necrosis and regeneration, and often proliferation of connective and adipose tissue. The progressive muscular dystrophies include at least Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), Limb-Girdle muscular dystrophies, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), Myotonic dystrophy (Steinert's disease) and congenital muscular dystrophies.
Currently there is no cure for these diseases, but certain medications and therapies have been shown to be effective. For instance, corticosteroids have been shown to slow muscle destruction in Duchene muscular dystrophy patients. While corticosteroids can be effective in delaying progression of the disease in many patients, long-term corticosteroid use is undesirable due to unwanted side effects.
Researchers are also investigating the potential of certain muscle-building medicines. One such approach is to block the protein myostatin, a growth factor known to play a role in the growth and development of muscle. For instance, monoclonal antibodies specific to myostatin have been shown to improve the condition of mice with muscular dystrophy, presumably by blocking the action of myostatin. The myostatin-blocking approach presents concerns however. For instance, blocking myostatin could interfere with the satellite cells that help replace injured or dead muscle cells. It is believed that myostatin helps keep satellite cells at rest until they are needed and, without myostatin, the satellite cells could become depleted. In addition, it has been proposed that myostatin blockers may be too targeted to boost muscle growth, as there are a variety of proteins similar to myostatin that also limit muscle growth
PCT Application No. WO 2007/059612 (Rudnicki et al.) describes a novel population of Pax7+/Myf5− satellite stem cells. This group was the first to confirm that satellite stem cells are a heterogeneous population containing stem cells (Pax7+/Myf5−) and progenitor cells (Pax7+/Myf5+). Prior to this disclosure, it was unclear whether satellite cells were stem cells, committed progenitors or de-differentiated myoblasts, and whether the niche was homogenous or heterogeneous. Using Cre/LoxP lineage-tracing, the group identified a sub-population of satellite cells which had never expressed Myf5 and functioned as a stem cell reservoir (see also Kuang et al., 2007). The group successfully isolated the Pax7+/Myf5− satellite stem cells, which were found to represent about 10% of the adult satellite cell pool and give rise to daughter satellite myogenic cells (Pax7+/Myf5+) through asymmetric apical-basal cell divisions. Transplantation of both Myf5− and Myf5+ FACS-sorted satellite cells demonstrated that satellite stem cells are capable of repopulating the adult satellite cell niche as well as self-renewal (Kuang et al., 2007). It has recently been demonstrated that, during skeletal muscle regeneration, the satellite cell population is maintained by the stem cell subpopulation, thus allowing tissue homeostasis and multiple rounds of regeneration during the lifespan of an individual (Kuang et al., 2007). Knowledge of the molecular networks regulating satellite stem cell fate decisions has remained unclear.
PCT Application No. WO 2004/113513 (Rudnicki et al.) discloses methods and compositions for modulating proliferation or lineage commitment of an atypical population of CD45+Sca1+ stem cells, located outside the satellite stem cell compartment, by modulating myogenic determination of Wnt proteins.
The Wnt family of genes encode over twenty cysteine-rich, secreted Wnt glycoproteins that act by binding to Frizzled (Fzd) receptors on target cells. Frizzled receptors are a family of G-protein coupled receptor proteins. Binding of different members of the Wnt-family to certain members of the Fzd family can initiate signaling by one of several distinct pathways. In the termed canonical pathway, activation of the signaling molecule, Disheveled, leads to the inactivation of glycogen synthase kinase-3 (GSK-3β), a cytoplasmic serine-threonine kinase. The GSK-3β target, β-catenin, is thereby stabilized and translocates to the nucleus where it activates TCF (T-cell-factor)-dependant transcription of specific promoters (Wodarz, 1998, Dierick, 1999). In the non-canonical, or planar cell polarity (PCP) pathway, binding of Wnt to Fzd also activates Disheveled, which in this case activates RhoA, a small g protein. Activation of the PCP pathway does not result in nuclear translocation of β-catenin.
Wnt signaling plays a key role in regulating developmental programs through embryonic development, and in regulating stem cell function in adult tissues (Clevers, 2006). Wnts have been demonstrated to be necessary for embryonic myogenic induction in the paraxial mesoderm (Borello et al., 2006; Chen et al., 2005; Tajbakhsh et al., 1998), as well in the control of differentiation during muscle fiber development (Anakwe et al., 2003). Recently, the Wnt planar cell polarity (PCP) pathway has been implicated in regulating elongation of differentiating myocytes in the developing myotome (Gros et al., 2009). In the adult, Wnt signaling is necessary for the myogenic commitment of adult CD45+/Sca1+ stem cells in muscle tissue following acute damage (Polesskaya et al., 2003; Torrente et al., 2004). Other studies suggest that canonical Wnt/β-catenin signaling regulates myogenic differentiation through activation and recruitment of reserve myoblasts. In addition, Wnt/β-catenin signaling in satellite cells within adult muscle appears to control myogenic lineage progression by limiting Notch signaling and thus promoting differentiation. Thus, traditionally, it has been assumed that Wnt proteins act as stem cell growth factors, promoting the proliferation and differentiation of stem cells and/or progenitor cells.
Stem cells, and therapies targeting stem cells, have the potential for providing benefit in a variety of clinical settings. A limitation to many potential therapeutic applications has been obtaining a sufficient number of undifferentiated stem cells, and stimulating terminal differentiation into mature tissue-specific cells without depleting the stem cell reservoir. Much current stem cell research focuses on directing the proliferation and differentiation of stem cells, in particular, transient amplifying progenitors, to repair or regenerate damaged tissue. In addition to concerns about stem cell depletion, another concern with stimulating proliferation and differentiation of stem cells is abnormal or poorly-formed tissue. Accordingly, there is a need in the art for continued research and development in the area of stem cells and for new and improved methods and compositions for modulating stem cell function in a physiological manner.